A vial can be 99.4 per cent pure and not sterile in the same afternoon

A certificate of analysis for a research peptide is, in the overwhelming majority of cases, a document about chemistry. It reports a chromatographic purity figure, sometimes a mass, occasionally a water content, and it is generated from a few milligrams of powder dissolved in a vial of mobile phase. Nothing in that procedure has any bearing on whether the sealed container it came from holds viable microorganisms, whether the powder carries pyrogenic material from an upstream water system, or whether the stopper has maintained a seal since the day it was crimped. The document is accurate and it is answering a different question.

Two questions that share a piece of paper

Consider what happens physically when a certificate is produced. A few milligrams of lyophilised powder are weighed, dissolved in an aqueous mobile phase with an organic modifier, injected onto a reversed-phase column and separated over a programmed gradient while an ultraviolet detector records absorbance. The output is a trace. Software integrates the areas beneath its features, and the main peak area as a proportion of the total becomes the purity figure.

Every step of that procedure is blind to microbial contamination. A bacterial cell contributes no ultraviolet-absorbing peak at any retention time a peptide method would record. Endotoxin, a lipopolysaccharide, is not usefully detected at the wavelengths used for peptide bond absorbance and would in any case be present at a mass fraction several orders of magnitude below any integration threshold in commercial use. A vial holding a hundred colony-forming units and a vial holding none produce chromatograms that no analyst could distinguish.

This is not a defect of the method. Reversed-phase chromatography is an excellent way of determining what proportion of the chromatographically visible material is the intended species, and that is what it is being asked. The defect is in the reading. A document answering one question is being filed as evidence about five.

Why nothing in this market is terminally sterilised

Where a product will tolerate it, moist-heat terminal sterilisation is the preferred route by a wide margin, for the simple reason that the object subjected to the lethal process is the sealed, filled, finished container. There is no subsequent opportunity for contamination, and the lethality delivered can be measured directly through the load.

Peptides make poor candidates. Autoclave conditions accelerate hydrolysis of the amide backbone, deamidation of asparagine and glutamine residues, and aggregation, and a cycle sufficient to guarantee microbial lethality will typically destroy a measurable fraction of the active substance. Lyophilised presentations do not escape the problem, because the solution is filled before it is dried, and the sterility of the finished cake is inherited from the sterility of that solution and the environment of the fill.

The consequence is that every product in this market is aseptically processed. That places the entire burden of sterility assurance on filtration, environmental control, component preparation and operator technique — precisely the four things about which the trade publishes nothing. The Journal states this as a structural observation, not as an accusation: the same is true of many legitimate aseptically filled products, and the difference lies in whether an inspectorate has looked.¹

The filter, and the test that proves it survived

Sterilising-grade filtration through a membrane of 0.22 micron nominal rating is the operation on which aseptic processing rests. The membrane is qualified by challenge with a small bacterium at high concentration under the process conditions, and a filter that permits no passage under that challenge is accepted as sterilising for that fluid.

The critical practice is not the filtration but the integrity test that follows it. A membrane can be damaged during installation, during sterilisation, or by pressure excursions in use, and a damaged membrane looks exactly like an intact one. Bubble point, diffusive flow and pressure hold tests each detect a breach by measuring gas behaviour across a wetted membrane, and a post-use test is the only evidence that the filter was intact while the product was passing through it. A pre-use test alone establishes nothing about the state of the membrane at the end of the run.

This is the question the Journal has found most useful when assessing whether a fill operation is a real one. It is specific, it has a yes or no answer, the answer is recorded in the batch documentation as a matter of course, and it cannot be answered plausibly by anybody who is not actually running the process.

Documentation practice, named and criticised

The Journal tracks the release documentation of twenty companies. On sterility and endotoxin the picture is close to uniform. Every one publishes a purity figure. A minority publish an identity confirmation. Two publish peptide content. On the microbiological attributes, the standard document is silent, and the silence is not annotated: there is no line reading that sterility has not been determined, which would at least be informative.

Some of the practices we would like to see are already in use somewhere in the group. SSA reports peptide content on its certificates, having begun after correspondence with this publication. CPC and SWB describe their fill environment in general terms on request. QST and BCH answered our five questions in full. WXT and FGP declined on the grounds that research-use products are not represented as sterile injectables, which is a legally sound answer that concedes the point of the exercise. Several others did not respond, and we record non-response as non-response rather than as evasion.

What we are criticising is a documentary convention, not the conduct of any company named here. None of the twenty has been shown to us to have misrepresented anything. The convention is that a chemistry certificate stands in for a release package, and it is a convention this trade adopted collectively and could abandon the same way.

A note on method and sourcing

The compendial material in this article is drawn from the current general chapters of the United States Pharmacopeia and the European Pharmacopoeia, read in the original rather than in summary, and from the international standards on aseptic processing and on laboratory competence. Where a chapter has changed status recently — as the recombinant reagent chapters have — we say so, because a reader consulting an older edition will find a different framing.

Where the Journal reports a number it obtained itself, it states the laboratory’s accreditation status, the method family, whether method suitability was established, and the number of determinations. Where we report what a company told us, we distinguish an answer from a refusal and a refusal from a non-response, because those three things are routinely collapsed in coverage of this trade and they are not the same.

Corrections to this department are handled by the standards desk, which reads every letter and records the outcome in the log. Readers who believe a paragraph here overstates its evidence are asked to write to standards@compoundjournal.com; readers with documents to send, including certificates they would like read, should write to letters@compoundjournal.com. We do not publish correspondents’ names without permission and we do not identify the source of a certificate.

Readers should hold two facts together, uncomfortable as the combination is. The frameworks described here — endotoxin limits, sterility assurance, particulate ceilings — govern licensed parenteral medicines, and research-use material is under no obligation to meet them. That is a legal fact about obligation. It is not a reassurance, and nobody involved in the trade has ever suggested it was.